DIVERGENT EFFECTS OF ATP ON THE BINDING OF THE DNAK AND DNAJ CHAPERONES TO EACH OTHER, OR TO THEIR VARIOUS NATIVE AND DENATURED PROTEIN SUBSTRATES

Using the native proteins lambda P, lambda O, sigma(32), and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we s how that DnaK and DnaJ molecular chaperones possess differential affin ity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, w hich are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA pro tein. The native sigma(32) transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactal bumin or native lambda P binds more efficiently to DnaK than to the Dn aJ molecular chaperone. The presence of nucleotides does not change th e DnaJ affinity to any of the tested protein substrates. In the case o f DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP anal ogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonh ydrolyzable ATP analogue inhibits the DnaK DnaJ complex formation. Mor eover, the DnaKc94 mutant protein lacking 94 amino acids from its C te rminal domain, which still possesses the ATPase activity and forms a t ransient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolys is, possesses low affinity to the protein substrates but can efficient ly interact with DnaJ molecular chaperone.